Cells
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2022
·Vol. 11
·期 22
·3689
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IF 7.666
Hao Liu,Jun-Tao Zhang,Chen-Ye Mou,Yue Hao,Wei Cui
Background: D-amino acid oxidase (DAAO) is a flavoenzyme that specifically catalyzes the deamination of many neutral and basic D-amino acids. This study aims to explore the pathological increment of hippocampal DAAO and its potential relationship with delayed hippocampal neuronal death. Methods: Ischemia–reperfusion was induced in mice through middle cerebral artery occlusion (MCAO). Neurological deficit scores and hippocampal neuronal death were assessed in MCAO mice. Immunofluorescent staining was applied to identify activated astrocytes and evaluate DAAO expression. TUNEL and Nissl staining were utilized to identify cell apoptosis of hippocampal neurons. Results: Hippocampal astrocytic DAAO was strikingly increased following ischemic stroke, with the greatest increase on day 5 after surgery, followed by the manifestation of neurobehavioral deficits. Astrocytic DAAO was found to be mainly expressed in the hippocampal CA2 region and linked with subsequent specific neural apoptosis. Thus, it is supposed that the activation of astrocytic DAAO in ischemic stroke might contribute to neuronal death. An intravenous, twice-daily administration of 4H-furo[3,2-b]pyrrole-5-carboxylic acid (SUN, 10 mg/kg) markedly relieved behavioral status and delayed hippocampal neuronal death by 38.0% and 41.5%, respectively, compared to the model group treated with saline. In transfected primary astrocytes, DAAO overexpression inhibits cell activity, induces cytotoxicity, and promotes hippocampal neuronal death at least partly by enhancing H2O2levels with subsequent activation of TRP calcium channels in neurons. Conclusions: Our findings suggest that increased hippocampal DAAO is causally associated with the development of delayed neuronal death after MCAO onset via astrocyte–neuron interactions. Hence, targeting DAAO is a promising therapeutic strategy for the management of neurological disorders.Keywords:D-amino acid oxidase;middle cerebral artery occlusion;neuronal death;transient receptor potential channel;hydrogen peroxide
研究领域:
神经科学
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药理学
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细胞生物学
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产品关联
DOI
PubMed
高影响因子
BRITISH JOURNAL OF PHARMACOLOGY
·
2019
·Vol. 176
·期 17
·3336-3349
·
IF 6.583
Hao Liu,Meng-Jing Zhao,Zi-Ying Wang,Qiao-Qiao Han,Hai-Yun Wu,Xiao-fang Mao,Yong-Xiang Wang
Background and Purpose d -Amino acid oxidase (DAAO) is a flavine adenine dinucleotide-containing flavoenzyme and specifically catalyses oxidative deamination of d -amino acids. This study aimed to explore the association between increased cerebral DAAO expression or enzymic activity and the development of cerebral ischaemia. Experimental Approach A mouse model of transient (90 min) middle cerebral artery occlusion (MCAO) was established, and western blotting, enzymic activity assay, and fluorescent immunostaining techniques were used. Key Results The expression and enzymic activity of DAAO increased over time in the cortical peri-infarct area of the mice subjected to transient MCAO. The DAAO was specifically expressed in astrocytes, and its double immunostaining with the astrocytic intracellular marker, glial fibrillary acidic protein, in the cortical peri-infarct area was up-regulated following ischaemic insult, with peak increase on Day 5 after MCAO. Single intravenous injection of the specific and potent DAAO inhibitor Compound SUN reduced the cerebral DAAO enzymic activity and attenuated neuronal infarction and neurobehavioural deficits with optimal improvement apparent immediately after the MCAO procedure. The neuroprotective effect was dose dependent, with ED 50 values of 3.9–4.5 mg·kg −1 . Intracerebroventricular injection of the DAAO gene silencer siRNA/DAAO significantly reduced cerebral DAAO expression and attenuated MCAO-induced neuronal infarction and behavioural deficits. Conclusions and Implications Our results, for the first time, demonstrated that increased cerebral astrocytic DAAO expression and enzymic activity were causally associated with the development of neuronal destruction following ischaemic insults, suggesting that targeting cerebral DAAO could be a potential approach for treatment of neurological conditions following cerebral ischaemia.
研究领域:
神经科学
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药理学
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分子生物学
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神经病学
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产品关联
DOI
PubMed
高影响因子
ADVANCED SYNTHESIS & CATALYSIS
·
2019
·Vol. 361
·期 13
·3191-3199
·
IF 5.451
Shuyun Ju,Mingxin Qian,Gang Xu,Lirong Yang,Jianping Wu
Optically pure 1,2,3,4-tetrahydroisoquinoline carboxylic acids constitute an important class of building blocks for the synthesis of natural products and synthetic pharmaceuticals. However, redox deracemization of racemic 1,2,3,4-tetrahydroisoquinoline carboxylic acids as an attractive method is still challenging for the lack of suitable oxidoreductases. Herein, a D-amino acid oxidase from Fusarium solani M-0718 ( Fs DAAO) with broad substrate scope and excellent enantioselectivity was exploited through genome mining, and applied for the kinetic resolution of a number of racemic 1- and 3-carboxyl substituted tetrahydroisoquinolines to yield the corresponding ( S )-enantiomers with excellent enantiomeric excess ( ee ) values (up to >99%). By using Fs DAAO in combination with ammonia-borane in one pot, deracemization of these racemic carboxyl-substituted tetrahydroisoquinolines was achieved with conversions up to >98% and >99% ee . Preparative-scale deracemization of racemic 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid and 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid was also demonstrated with good isolated yields (82% and 73%, respectively) and ee >99%. Our study provides an effective method for the synthesis of enantiomeric pure 1,2,3,4-tetrahydroisoquinoline carboxylic acids. This method is expected to provide access to chiral carboxyl-substituted 1,2,3,4-tetrahydroquinolines and 1,2,3,4-tetrahydro-ß-carbolines.
研究领域:
生物化学
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有机合成
·
酶工程
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产品关联
DOI
高影响因子
BIOTECHNOLOGY AND BIOENGINEERING
·
2023
·
IF 3.8
Liuyu Wang,Heng Tang,Hongli Zhu,Yaping Xue,Yuguo Zheng
D -Amino acid oxidase (DAAO) selectively catalyzes the oxidative deamination of D -amino acids, making it one of the most promising routes for synthesizing optically pure L -amino acids, including L -phosphinothricin ( L -PPT), a chiral herbicide with significant market potential. However, the native DAAOs that have been reported have low activity against unnatural acid substrate D -PPT. Herein, we designed and screened a DAAO from Rhodotorula taiwanensis ( Rtw DAAO), and improved its catalytic potential toward D -PPT through protein engineering. A semirational design approach was employed to create a mutation library based on the tunnel-pocket engineering. After three rounds of iterative saturation mutagenesis, the optimal variant M 3rd -SHVG was obtained, exhibiting a >2000-fold increase in relative activity. The kinetic parameters showed that M 3rd -SHVG improved the substrate binding affinity and turnover number. This is the optimal parameter reported so far. Further, molecular dynamics simulation revealed that the M 3rd -SHVG reshapes the tunnel-pocket and corrects the direction of enzyme–substrate binding, allowing efficiently catalyze unnatural substrates. Our strategy demonstrates that the redesign of tunnel-pockets is effective in improving the activity and kinetic efficiency of DAAO, which provides a valuable reference for enzymatic catalysis. With the M 3rd -SHVG as biocatalyst, 500 mM D, L -PPT was completely converted and the yield reached 98%. The results laid the foundation for further industrial production.
研究领域:
生物化学
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酶学
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蛋白质工程
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生物催化
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分子动力学
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产品关联
DOI
PubMed
高影响因子
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